Leonid A. Serebryannyy

Nuclear actin and myosins: Life without filaments

Actin and myosin are major components of the cell cytoskeleton, with structural and regulatory functions that affect many essential cellular processes. Although they were traditionally thought to function only in the cytoplasm, it is now well accepted that actin and multiple myosins are found in the nucleus. Increasing evidence on their functional roles has highlighted the importance of these proteins in the nuclear compartment.

α-Catenin is an inhibitor of transcription.

Significance Recent studies have shown that actin-binding and -regulating proteins, originally characterized in the context of cytoskeletal events, can also modify gene expression through directly impacting actin-dependent transcription. This study shows α-catenin (α-cat), an actin-binding protein that is essential for cell–cell adhesion and contact-dependent growth inhibition, can antagonize Wnt/β-catenin–mediated transcription and impact nuclear actin properties, suggesting that these events may be related. These findings establish α-cat as one of a growing list of actin-binding proteins that can modulate transcription, possibly by controlling actin dynamics in the nucleus. α-Catenin (α-cat) is an actin-binding protein required for cell–cell cohesion. Although this adhesive function for α-cat is well appreciated, cells contain a substantial amount of nonjunctional α-cat that may be used for other functions. We show that α-cat is a nuclear protein that can interact with β-catenin (β-cat) and T-cell factor (TCF) and that the nuclear accumulation of α-cat depends on β-cat. Using overexpression, knockdown, and chromatin immunoprecipitation approaches, we show that α-cat attenuates Wnt/β-cat–responsive genes in a manner that is downstream of β-cat/TCF loading on promoters. Both β-cat– and actin-binding domains of α-cat are required to inhibit Wnt signaling. A nuclear-targeted form of α-cat induces the formation of nuclear filamentous actin, whereas cells lacking α-cat show altered nuclear actin properties. Formation of nuclear actin filaments correlates with reduced RNA synthesis and altered chromatin organization. Conversely, nuclear extracts made from cells lacking α-cat show enhanced general transcription in vitro, an activity that can be partially rescued by restoring the C-terminal actin-binding region of α-cat. These data demonstrate that α-cat may limit gene expression by affecting nuclear actin organization.

A role for nuclear actin in HDAC 1 and 2 regulation

Class I histone deacetylases (HDACs) are known to remove acetyl groups from histone tails. This liberates positive charges on the histone tail and allows for tighter winding of DNA, preventing transcription factor binding and gene activation. Although the functions of HDAC proteins are becoming apparent both biochemically and clinically, how this class of proteins is regulated remains poorly understood. We identified a novel interaction between nuclear actin and HDAC 1 and HDAC 2. Nuclear actin has been previously shown to interact with a growing list of nuclear proteins including chromatin remodeling complexes, transcription factors and RNA polymerases. We find that monomeric actin is able to bind the class I HDAC complex. Furthermore, increasing the concentration of actin in HeLa nuclear extracts was able to suppress overall HDAC function. Conversely, polymerizing nuclear actin increased HDAC activity and decreased histone acetylation. Moreover, the interaction between class I HDACs and nuclear actin was found to be activity dependent. Together, our data suggest nuclear actin is able to regulate HDAC 1 and 2 activity.

Persistent nuclear actin filaments inhibit transcription by RNA polymerase II

ABSTRACT Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin–RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II. Highlighted Article: Depletion of actin in the nucleus by polymerization into stable nuclear actin filaments inhibits the actin–polymerase-II interaction, resulting in impaired RNA polymerase II activity, localization, and gene recruitment.

Nuclear actin and myosins in adenovirus infection.

Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the hosts transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus.

Tumor stiffness is unrelated to myosin light chain phosphorylation in cancer cells.

Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.

Nuclear α-catenin mediates the DNA damage response via β-catenin and nuclear actin

ABSTRACT α-Catenin is an F-actin-binding protein widely recognized for its role in cell–cell adhesion. However, a growing body of literature indicates that α-catenin is also a nuclear protein. In this study, we show that α-catenin is able to modulate the sensitivity of cells to DNA damage and toxicity. Furthermore, nuclear α-catenin is actively recruited to sites of DNA damage. This recruitment occurs in a β-catenin-dependent manner and requires nuclear actin polymerization. These findings provide mechanistic insight into the WNT-mediated regulation of the DNA damage response and suggest a novel role for the α-catenin–β-catenin complex in the nucleus. Highlighted Article: The nuclear α-catenin–β-catenin complex mediates DNA damage sensitivity in a WNT-dependent manner. α-Catenin recruitment to sites of DNA damage is dependent on β-catenin and nuclear actin.

The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology.